Use of 4-[(4-thiazolyl)phenoxy]alkoxy-benzamidine derivatives for treatment of osteoporosis

ABSTRACT

This invention relates to a pharmaceutical composition containing 4-[(4-thiazolyl)phenoxyl]alkoxy-benzamidine derivatives expressed by the following formula 1 for the prevention and treatment of osteoporosis and more particularly, to the use of 4-{5-[4-(5-isoproply-2-methyl-1,3-thiazol-4-yl)phenoxyl]pentoxy}-benzamidine or N-hydroxy-4-{5-[4-(5-iso-propyl-2-methyl-1,3-thiazol-4-yl) phenoxyl]pentoxy}-benzamidine expressed by the following formula 1, which is known as an antagonist of leukotriene-B 4  receptor, as a pharmaceutical composition for the prevention and treatment of osteoporosis. [Formula 1], wherein, R is a hydrogen atom or a hydroxy group.

TECHNICAL FIELD

[0001] The present invention relates to a pharmaceutical compositioncontaining 4-[(4-thiazolyl)-phenoxy]alkoxy-benzamidine derivativesrepresented by the following formula 1 for the prevention and treatmentof osteoporosis and more particularly, to the pharmaceutical compositioncontaining4-{5-[4-(5-isopropyl-2-methyl-1,3-thiazol-4-yl)phenoxy]pentoxy}-benzamidine(hereinafter referred to as “DW1352”) orN-hydroxy-4-{5-[4-(5-isopropyl-2-methyl-1,3-thiazol-4-yl)phenoxy]pentoxy}-benzamidine(hereinafter referred to as “DW1350”) represented by the followingformula 1, which is reported to have leukotriene-B₄ (hereinafterreferred to as “LTB-4”) receptor antagonism for the prevention andtreatment of osteoporosis.

[0002] Wherein, R is a hydrogen atom or a hydroxy group.

BACKGROUND ART

[0003] Bone is the structural material of the body's framework andserves to maintain the necessary bone mass and structure. Bone containscalcium (Ca²⁺) and plays an important role in maintaining the calciumlevel in the blood. To this end, the growth of bone is a metabolicbalance between the activity of osteoblasts and osteoclasts in the boneremodelling cycle.

[0004] When the balance between bone absorption and bone formation isdisrupted, the amount of bone tissue replaced by osteoblasts fails tomatch that absorbed by osteoclasts, thus leading to osteoporosis, acommon condition to cause loss of bone density or bone mass. Thisdisease is frequently occurring in middle-aged or elderly women.

[0005] To date, the established strategy has been to produce drugscapable of preventing bone loss by inhibiting osteoclastic boneabsorption. Attempts to develop alternative therapies, such as LTB-4receptor antagonist, have been made but their development towardseffective anti-osteoporotic agent has been unsuccessful due toinsufficient inhibition on osteoclastic bone absorption. Therefore,there is an urgent need for new osteoporosis therapies aimed atsuppressing osteoclastic bone absorption.

[0006] 4-[(4-thiazolyl)-phenoxy] alkoxy-benzamidine derivative, togetherwith its process for preparation, has been already known asleukotriene-B₄ receptor antagonist (Lee Sung-eun, Synthesis andBiological Activity of Natural Products and Designed New HybridCompounds for the Treatment of LTB ₄ Related Disease, Ph.D thesis,Graduate School of Pusan Univ., August 1999).

[0007] The natural product LTB-4 is one of arachidonate metabolitesformed via 5-lipoxygenase pathway [Ford-Hutchinson, A. W. et al., Nature(London), 286, 264-265, 1980].

[0008] The recent studies have focused on the influence of arachidonatemetabolites on the bone tissue metabolism.

[0009] 5-lipoxygenase metabolites produced from osteoblasts are found tostimulate bone absorption (Meghji, S. et. al., Calcif. Tiss. Int. 36,139-149, 1988); the interstitial cells C433 obtained from a giant celltumor is involved in producing 5-lipoxygenase metabolites to increasethe counts and activity of osteoblasts (Mundy, G. R., J. Bio. Chem. 268,10087-10094, 1993); the bone absorption function may be stimulated withthe addition of synthetic LTB-4 during the cultivation process of bonetissue (Bonewald, L. F., J. Bone Miner. Res. 11, 521-529, 1996); andBoth in vitro and in vivo studies have demonstrated that LTB4 inducesthe bone absorption via production of osteoclasts (Bonewald, L. F., JBone Miner. Res. 11, 1619-1627, 1996).

[0010] Currently, many studies have been under way with the conceptionthat some compound showing an antagonistic action against LTB-4 receptormay affect the embolic diseases of bone tissue.

[0011] The inventors have conducted intensive studies to identify anumber of diverse-structure compounds useful as effective LTB4 receptorantagonists, aimed at suppressing osteoclastic bone absorption orstimulating osteoblastic bone formation. In consequence it has beenidentified that 3-amino-1,2-benzoisoxazole derivative represented by thefollowing formula 2 is effective in the prevention and treatment ofosteoporosis, while exerting antagonistic action against LTB4 receptor.The inventors filed a patent application of such compound dated Feb. 4,1998(KR 98-3138).

[0012] Wherein, n is an integer of 3˜5.

[0013] In an effort to identify alternative osteoporosis therapies, theinventors have tested the inhibitory action of4-[(4-thiazolyl)-phenoxy]alkoxy-benzamidine derivatives as LTB-4receptor antagonist; among these derivatives, such compound as4-{5-[4-(5-isopropyl-2-methyl-1,3-thiazol-4-yl)phenoxy]pentoxy}-benzamidineorN-hydroxy-4-{5-[4-(5-isopropyl-2-methyl-1,3-thiazol-4-yl)phenoxy]pentoxy}-benzamidineis found to be significantly effective in preventing bone loss byinhibiting osteoclastic bone absorption. Thus, the present invention hasbeen finally completed.

DISCLOSURE OF THE INVENTION

[0014] The present invention relates to the therapeutic use of apharmaceutical composition containing4-{5-[4-(5-isopropyl-2-methyl-1,3-thiazol-4-yl)phenoxy}pentoxy}-benzamidineor N-hydroxy-4-{5-[4-(5-isopropyl-2-methyl-1,3-thiazol-4-yl)phenoxy]pentoxy}-benzamidine represented by the following formula 1 forthe prevention and treatment of osteoporosis.

[0015] Wherein, R is a hydrogen atom or a hydroxy group.

[0016] 4-[(4-thiazolyl)-phenoxy]alkoxy-benzamidine derivatives may beprepared by the conventional method (Lee Sung-eun, Synthesis andBiological Activity of Natural Products and Designed New HybridCompounds for the Treatment of LTB ₄ Related Disease, Ph.D thesis,Graduate School of Pusan Univ., August 1999). Compounds of the presentinvention represented by the formula 1 may be also used withpharmaceutically acceptable salts using the following materials:inorganic acids (hydrochloric acid, bromic acid, sulfuric acid andphosphoric acid); organic acids (citric acid, acetic acid, lactic acid,tartaric acid, fumaric acid, formic acid, propionic acid, oxalic acid,trifluoroacetic acid, methanesulfonic acid, benzoic acid, maleic acid,gluconic acid, glycollic acid, succinic acid, 4-toluenesulfonic acid,galacturonic acid, embonic acid, glutamic acid or aspartic acid.According to the present invention, it is preferred to employhydrochloric acid as inorganic acid and methanesulfonic acid as organicacid.

[0017] The anti-osteoporotic composition of the present invention may beapplied in a therapeutically effective dose via various routes ofadministration. Any person having an ordinary knowledge in the technicalfield to which the present invention belongs can determine any dosageform and dosing regimen depending on purpose of administration, routesof administration, severity of diseases and body weight.

[0018] The anti-osteoporotic composition of the present inventioncontains4-{5-[4-(5-isopropyl-2-methyl-1,3-thiazol-4-yl)phenoxy]pentoxy}-benzamidineorN-hydroxy-4-{5-[4-(5-isopropyl-2-methyl-1,3-thiazol-4-yl)phenoxy]pentoxy}-benzamidinerepresented by the following formula 1 and its pharmaceuticallyacceptable carriers. The pharmaceutically acceptable carriers mayinclude every type of standard pharmaceutical carriers used for theconventional dosage forms, such as sterile solution, tablet (includingcoated tablet) and capsules. The typical examples of such carrierinclude some excipients (e.g., starch, milk, sugar, specific clay,gelatin, stearic acid, talc, vegetable fat or oil, gum, glycols), orother conventional excipients. Such carriers may also include flavoringagents, color additives and other materials. The composition containingsuch carriers may be formulated by the conventional method.

[0019] The anti-osteoporotic composition of the present inventioncontaining4-{5-[4-(5-isopropyl-2-methyl-1,3-thiazol-4-yl)phenoxy]pentoxy}-benzamidineorN-hydroxy-4-{5-[4-(5-isopropyl-2-methyl-1,3-thiazol-4-yl)phenoxy]pentoxy}-benzamidineor its salts may be applied via the conventional routes ofadministration (e.g. oral, intravenous, intramuscular or transdermal)but not limited to these routes of administration.

[0020] A wide range of therapeutic doses of4-{5-[4-(5-isopropyl-2-methyl-1,3-thiazol-4-yl)phenoxy]pentoxy}-benzamidineorN-hydroxy4-{5-[4-(5-isopropyl-2-methyl-1,3-thiazol-4-yl)phenoxy]pentoxy}-benzamidinehas been established for the prevention and treatment of osteoporosis.The therapeutic dose level for the treatment of osteoporosis is 10˜1000mg daily. Any person having an ordinary knowledge in the technical fieldto which the present invention belongs can determine the dose and dosingfrequency depending on characteristics of an agent, severity of diseaseand body weight, size of inflammation and routes of administration.

BEST MODE FOR CARRYING OUT THE INVENTION

[0021] The present invention is explained in more detail by thefollowing examples.

EXAMPLE 1 Inhibitory Effects on Osteoclast Differentiation of Each TestSubstance

[0022] The effect of each test substance on osteoclast proliferation anddifferentiation process were evaluated via co-culture with osteoblast.

[0023] 1. Preparation of Cells

[0024] a) Preparation of Bone Marrow Cells

[0025] Tibia and Femora were aseptically ectomized from male ddY mice of6˜8 weeks to harvest bone marrow cells by using a syringe (21G, KoreaGreen Cross).

[0026] The bone marrow cells were suspended in 5 mL α-MEM medium (GibcoBRL Co.) containing sodium bicarbonate (2.0 g/L), streptomycin (100mg/L) and penicillin (100,000 unit/mL). The harvested cells werecentrifuged at 800×g for 5 mins to collect the whole quantity. To removethe red blood cells within bone marrow cells, 3 mL of Tris HCl (0.83%NH₄Cl, pH7.5) was added and well mixed. After centrifuging above cells,the numbers of bone marrow cells were counted and then, the bone marrowcells were immediately used for co-culture system with osteoblast.

[0027] b) Preparation of osteoblast

[0028] The calvaria were aseptically ectomized from neonate ICR mice of1˜2 days, washed with PBS solution and incubated with a mixture ofenzyme solution (0.2% collagenase and 0.1% dispase) at 37° C. gentleshaker. This procedure was sequentially repeated (10, 10, 10, 20, 20 and20 mins), and then the calvaria cells having the characteristics ofosteoblast, were mostly released from III˜VI digestion groups, werecollected and washed with the medium (serum-free α-MEM). The washedcells were cultivated in α-MEM medium containing 10% FBS for 2˜3 days.After subculturing, these cells were used for this experiment, anddiluted to reach the concentration of 1×10⁶ cells/mL for storage at −70°C.

[0029] 2. Measurement of Osteoclast Differentiation

[0030] a) Preparation of Specimen

[0031]N-hydroxy-4-{5-[4-(5-isopropyl-2-methyl-1,3-thiazol-4-yl)phenoxy]pentoxy}-benzamidine(DW1350) and4-{5-[4-(5-isopropyl-2-methyl-1,3-thiazol-4-yl)phenoxy]pentoxy}-benzamidine(DW1352) used for test substances of the present invention, andN,N-diisopropyl4-[4-(3-aminobenzo[d]isooxazole-6-yloxy)butoxy]-3-methoxybenzamide(hereinafterreferred to as “HS-1141”) and4-[5-[4-(aminoiminomethyl)phenoxy]pentoxy]-3-methoxy-N,N-bis(1-methylethyl)benzamidemaleic acid (Morrissey, M. M., Suh, H. U.S. Pat. No. 5,451,700;hereinafter referred to as “CGS-25019C”), LTB-4 receptor antagonists ascontrol, were dissolved in a sterile distilled water to make desiredconcentrations following dilution. The volume of final specimen added tothe medium was determined at the ratio of 1:1000.

[0032] b) Reaction with Specimens Via Co-Culture System

[0033] Bone marrow cells, so prepared from the above No. 1, andosteoblast from calvaria were co-cultured for osteoclastdifferentiation. Both bone marrow cells (25,000 cells/cm²) andosteoblast (10,000 cells/cm²) were plated on a 96 well plate in α-MEMmedium containing 10% FBS with specimen, and then culture the reactionmixture for 7 days. Some differentiation factors, such as dexamethasone(10⁻⁶M) and vitamin D₃ (10⁻⁹M), were also continuously added to themedium from the first day of cultivation. The media were changed withfresh media containing a mixture of specimens and differentiationfactors every 2˜3 day.

[0034] c) Evaluation of Osteoclast Differentiation

[0035] 1) Preparation of Tartarate Resistance Acid Phosphatase (TRAP)Staining Solution

[0036] TRAP was used as a marker to measure osteoclast in considerationof its characteristics showing a positive reaction to TRAP stainingsolution. TRAP staining solution was prepared in a manner such that 5 mgof naphtol AS-MS phosphate (sigma N4875), a substrate and 25 mg ofcoloring agent (Fast Red Violet LB salt) was dissolved inN,N-dimethylformamide (about 0.5 mL) and with the addition of 0.1NNaHCO₃ buffer solution (50 mL) containing 50 mM of tartaric acid, thereaction mixture was stored at refrigerator prior to use.

[0037] 2) Staining Method

[0038] After 7-day culture, the medium was removed from the wells andthen, the cells were once washed with PBS solution and fixed to PBScontaining 10% formalin for 2˜5 mins. The cells were also fixed in amixed solution, ethanol and acetone (1/1), for about 1 min, and driedoff. The cells were further treated by TRAP staining solution for 15mins and washed with PBS to measure the experimental results with thestaining degree of cells under a microscopic examination.

[0039] 3) Analysis on the Experimental Results.

[0040] The counts of osteoclast only with more than 3 nuclei showing theTRAP-positive reaction were calculated under a microscopic examination,and each of test was reconfirmed over three times for gaining morereliable data.

[0041] As shown in the following table 1, the inhibitory effect of eachexperimental group on the differentiation of osteoclast versus controlswere expressed by inhibitory percentage value, and 50% inhibitoryconcentration on osteoclast differentiation was calculated as IC₅₀.

[0042] The anti-osteoporotic effect of each test substance were comparedwith controls, such as CGS-25019C and HS-1141 (U.S. Pat. No. 6,150,390and Korea Patent Application No. 98-3138), a conventionalanti-osteoporotic agent belonging to the same member of CGS-25019C,which demonstrates the antagonistic action to the existing LTB-4receptor. TABLE 1 % inhibitory action Specimen 3.2 nM 16 nM 80 nM 400 nMIC₅₀ DW1350 1.0 68.8 82.3 88.0 19.87 nM DW1352 50.0 81.8 83.9 92.7  1.25nM HS-1141 1.2 3.0 12.0 23.5 — CGS-25019C −8.9 8.3 0.0 17.7 —

[0043] As shown in the table 1, the experimental results indicate thatthe inhibitory effect of both DW1350 and DW1352 against osteoclastproliferation and differentiation were significantly better than thoseof HS-1141 and CGS-25019C. These test substances, which affect theosteoclast differentiation at a low concentration, may prove to beeffective for the prevention and treatment of osteoporosis.

EXAMPLE 2 Fusion Assay

[0044] This assay is designed to evaluate the influences of each testsubstance in terms of osteoclast fusion during the differentiationprocess in which immature prefusion osteoclasts (pOC; osteoclaststruture with one more nuclei) were transformed into maturemultinucleated osteoclast (OCL) via cell to cell fusion (GreggWesolowski et al. Experimental Cell Research 219, 679-686, 1995).

[0045] 1. Preparation of Prefusion Osteoclast (LOC)

[0046] The prefusion osteoclast can be obtained via co-culture of bothbone marrow cells and osteoblast, so prepared from Example 1. Themixture of both osteoblast (about 5×10⁵ cells/plate) and bone marrowcells (about 1×10⁷ cells/plate) were co-cultured in a 100 mm culturedish. Some differentiation factors, such as dexamethasone (10⁻⁶M) andvitamin D₃ (10⁻⁹M), were added to the medium from the first day ofculture. The medium was changed with the fresh medium containingdifferentiation factors every 2 day.

[0047] Since a great number of the preclusion osteoclasts having one ormore nuclei in fusion process were formed during 4-day co-culture, thecells were separated co-cultivation after 4 days. The medium was removedfrom the cells and with the addition of 0.2% collagenase solution (4mL), the cells were incubated at 37° C. for 20 mins to separate theattachment cells. Since the majority of separated cells wereosteoblasts, all osteoblasts were washed with PBS solution two or threetimes for their complete removal.

[0048] After the remaining prefusion osteoclasts were separated viareaction for 20 mins with the addition of echistatin containing 10% BSA,the cells were harvested by centrifuge.

[0049] 2. Reaction of Fusion Experiment

[0050] The test substances diluted at each concentration were diluted atthe desired concentration in α-MEM medium (addition of 10% FBS) to loadthem into a 96-well microplate in a dose of 100 μL per well. Theosteoclastic monocytes, so separated from the preceding No. 1, wereplated on a 96-well microplate in a dose of 5×10³ cell/100 μL per welland cultured at 37° C. for 24 hrs, thus resulting in the osteoclastfusion successfully. In the case of specimen-free and positive controls,experiments were performed in the same manner as above. The positivecontrol use for this experiment includes4-{4-[4-(5-isopropyl-2-methyl-1,3-thiazol-4-yl)phenoxy]butoxy}-benzamidine(hereinafter referred to as “DW1351”) andN-hydroxy-4-{4-[4-(5-isopropyl-2-methyl-1,3-thiazol-4-yl)phenoxy]butoxy}-benzamidine(hereinafter referred to as “DW1349”) which have the similar chemicalstructure to HS-1141, CGS-25019C, DW1350 and DW1352.

[0051] 3. Measurement of Osteoclast Fusion and its Analysis

[0052] The medium was removed from the cells and then, the cells wereonce washed with PBS once and fixed to PBS solution containing 10%formalin for about 5 mins. The cells were again fixed to both ethanoland acetone (1/1) in a mixing solution for about 5 mins and dried off.The cells were further treated by TRAP staining solution for 15 mins andwashed with water to observe the cells under the microscope. TheTRAP-positive osteoclast counts, which were differentiated from monocyteto multinucleated cells (osteoclast having more than 10 nuclei) viafusion process, were measured.

[0053] The following table 2 shows the differences of measured cellcounts versus control as % inhibitory concentration. TABLE 2 Inhibitoryaction (%) Specimen 0.08 uM 0.4 uM 2 uM 10 uM IC₅₀ DW1350 4.50 25.6480.00 97.95 0.81 uM DW1352 5.13 24.72 87.18 98.97 0.74 uM HS-1141 2.112.31 15.71 36.29 — CGS-25019C 10.14 13.04 13.77 12.32 — DW1351 0.0 0.038 74 — DW1349 0.0 2.3 4.5 18 —

[0054] As shown in the table 2, the experimental results demonstratethat both DW1350 and DW1352 exerted the significant inhibitory effectssagainst osteoclast fusion (IC₅₀: 0.81 and 0.74 uM, respectively). Morespecifically, the inhibitory effects of both DW1350 and DW1352 againstosteoclast fusion makes it possible to prevent the mature osteoclastformation which will result in the significant inhibition ofosteoclast-dependent bone absorption. The control CGS-25019C showedlittle inhibitory effect against osteoclast fusion, irrespective of drugconcentrations. The inhibitory effect of HS-1141 against osteoclastfusion was lower than those of DW1350 and DW1352, although the formerwas dependent on drug concentrations. In the case of DW1349 and DW1351having extremely similar structure to DW1350 and DW1352, theirinhibitory effects against osteoclast fusion were significantly lowerthan DW1350 and DW1352, although the former was dependent on drugconcentrations like HS-1141.

[0055] Therefore, it is expected that among4-[(4-thiazolyl)phenoxy]alkoxy-benzamidine derivatives, both DW1350 andDW1352 may be developed as new anti-osteoporotic agents by effectivelyinhibiting mature osteoclast formation based on the inhibitory mechanismof osteoclast fusion.

EXAMPLE 3 Measurement of Bone Resorption (Pit Formation Assays)

[0056] The mature osteoclast (OCL) is mainly involved in removingmineral by bone resorption. This experiment is designed to measure theinhibitory effects of each test substance on the bone resorption ofosteoclast using ivory fragment (Eijiro Jimi et al. Endocrinology 137,p2187-2190, 1996).

[0057] 1. Preparation of Mature Osteoclast

[0058] a) Preparation of Collagen Gel Solution

[0059] The co-culture system with for both bone marrow cells andosteoblast was performed using a cultivation dish containing collagengel (cell matrix Type I-A). Collagen, 5-fold concentrated α-MEM mediumand 0.05M NaOH buffer solution (2.2% NaHCO₃, pH7.4) were mixed at theratio of 7:2:1 at a low temperature, and then storage at a lowtemperature. Then, 4 mL of the mixed solution was added to a 100 mmculture dish, applied evenly and left at 37° C. for 5 minutes.

[0060] b) Preparation of Mature Osteoclast Via Co-Culture System

[0061] Using α-MEM medium, the mixture of both bone marrow cells (about1×10⁷ cells/plate) and osteoblast (about 5×10⁵ cells/plate), soseparated from Example 1, were plated on a 100 mm dish containingcollagen gel. The co-culture was performed in the presence ofdifferentiation factors, such as vitamin D (10⁻⁹M) and dexamethasone(10⁻⁶M). As described above, a great number of mature multinucleatedosteoclasts with the ability of bone resorption were obtained via 7-dayco-culture. The medium was removed from the cells and with the additionof 0.2% collagenase solution, the attachment cells were separated byincubation for 20 minutes. The cells were collected via centrifuge. Theharvested crude osteoclasts were again diluted in α-MEM medium to makethe cells of 5,000 cells/100 μl.

[0062] 2. Preparation of Hematoxylin Staining Solution

[0063] Hematoxylin staining solution was prepared in a manner such thatmade hematoxylin (1 g) was dissolved in 500 ml of distilled water andwith the addition of 500 ml of distilled water and sodium iodide (0.2g), the reaction mixture was stirred for 15 mins. Ammonium alum (50 g)and 7.5 ml of acetic acid were further added to the reaction mixture andfiltered off.

[0064] 3. Reaction on Ivory Fragment

[0065] After the ivory fragments, so cut with a thickness of 1 mm, weresterilized, each fragment was placed into a 96 well plate and then, 100μl α-MEM medium (10% FBS) was added. To measure its inhibitory effectagainst the pit formation of osteoclast, each test substance was addedin a maximum amount of 3 μl per concentration. With the addition of testsubstances, 100 μl of osteoclast solution was further added, mixedvigorously and cultured using 5% CO₂ incubator at 37° C. for 24 hrs. Toobserve the pits formed on the ivory fragments, the portion of grownosteoclast was directed upward and placed on a paper towel afterremoving them from the 96 well plate. With the removal of cells on theivory, 10 μl of hematoxylin solution was dropped on the ivory to performthe staining for about 5 mins. The surface of ivory fragments was rubbedwith a soft cotton pole to completely remove the staining solution.

[0066] 4. Observation of Pits Formation and its Analysis

[0067] The following table 3 shows the number of pits on ivory fragmentversus control as an inhibition percentage at various concentrationunder a microscopic examination. TABLE 3 Inhibitory action (%) Specimen0.016 μM 0.08 μM 0.4 μM 2 μM 10 μM IC₅₀ DW1350 32.2 53.9 65.2 84.3 91.30.075 μM DW1352 25.0 48.7 61.3 81.7 90.0 0.131 μM HS-1141 9 33 50.4 75.388.7 0.421 μM CGS- 0 0 2 9.2 17.3 — 25019C

[0068] As shown in the table 3, the experimental results demonstratethat both DW1350 and DW1352 exerted the significant inhibitory effectagainst the bone resorption of osteoclast. It also reveals that DW1350and DW1352 had the IC₅₀ values of 0.075 μM and 0.131 μM, respectively,3˜6 times of inhibitory effect higher than HS-1141. In the case ofCGS-25019C, a positive control, had a low inhibitory effect against theosteoclastic bone resorption.

EXAMPLE 4 Evaluation of Alkaline Phosphatase (ALP) Activity to MeasureOsteoblast Activity

[0069] This experiment is designed to evaluate the differentiation andactivity of osteoblast via ALP activity having a close relationship withosteoblastic bone formation (Y. Wada et al., Bone, 22, 479-485, 1998).

[0070] MC3T3-E1 cells (3,000 cells/well) derived from osteoblast wereplaced on a 96 well plate and after 24-hour culture, the media werechanged with fresh medium containing various differentiation factorssuch as ascorbic acid (100 μg/ml) and β-glycerophosphatic acid (5 mM).The medium was also treated with test substances and the medium,containing differentiation factors and specimen, was changed with freshmedium every 3 days.

[0071] The culture was terminated after two weeks to measure ALPactivity. With the removal of supernatant, 0.5% Triton X-100 were addedfor the lysis of cells. 100 μl of p-nitrophenylphosphate (1.21 mM) wasadded to 50 μl of above mixture. The mixture was incubated at 37° C. for30 mins and with the addition of 0.2N sodium hydroxide (50 μl), thereaction was terminated. The standard curve was indicated at theabsorbance of 405 nm using p-nitrophenol as a standard material andthen, the absorbance of test substances, so reacted, was measured toobserve the production amount of p-nitrophenol.

[0072] As shown in the following table 4, the units of ALP activity weredetermined as the amount of p-nitrophenol (nM) produced per time (permin or hour)/1 μg protein after measuring the amounts of proteincontained in reaction mixture of each test substance. TABLE 4 Specimen(10⁻⁸ M) ALP activity (units) DW1350 19.8 DW1352 17.1 HS-1141 15.2CGS-25019C 15.0 Controls 13.5

[0073] As shown in the table 4, the experimental results demonstratethat DW1350 exerted the highest ALP activity among all test substances.The ALP activities of DW1352 were also found to be superior to those ofcontrols, HS-1141and CGS-25019C. This experiment has indicated that bothDW1350 and DW1352 were effective in stimulating osteoblast activity byaffecting osteblast differentiation and formation. Therefore, bothDW1350 and DW1352 are quite useful drugs for the prevention andtreatment of osteoporosis, since it can suppress the osteoclasticfunction, while stimulating the osteoblastic activity.

Industrial Applicability

[0074] The aforementioned examples have revealed that both DW1350 andDW1352, a LTB-4 receptor antagonist, exert better inhibitory effectagainst osteoclast in terms of differentiation, formation, fusion andbone absorption.

[0075] Both agents may prove to be effective for the prevention andtreatment of osteoporosis, since they can suppress the osteoclasticfunction with enhanced stimulation of osteoblastic activity, compared toDW1349 and DW1351 with the structural similarity, as well as HS-1141 andCGS-25019C.

[0076] Therefore, it is expected that the compound of the presentinvention may provide the basis for new osteoporosis therapies aimed atsuppressing the osteoclastic bone sorption and stimulating theosteoblastic bone formation, including the treatment of LTB-4 relateddiseases.

1. A preventive or therapeutic agent for osteoporosis comprising theeffective amount of the following formula 1 or its salts:

Wherein, R is a hydrogen atom or a hydroxy group.
 2. A preventive ortherapeutic agent for osteoporosis, according to claim 1, comprising theeffective amount of theN-hydroxy-4-{5-[4-(5-isopropyl-2-methyl-1,3-thiazol-4-yl)phenoxy]pentoxy}-benzamidineor its salts.
 3. A preventive or therapeutic agent for osteoporosis,according to claim 1, comprising the effective amount of the4-{5-[4-(5-isopropyl-2-methyl-1,3-thiazol-4-yl)phenoxy]pentoxy}-benzamidineor its salts.
 4. A preventive or therapeutic agent for osteoporosis,according to claim 2, comprising the effective amount of theN-hydroxy-4-{5-[4-(5-isopropyl-2-methyl-1,3-thiazol-4-yl)phenoxy]pentoxy}-benzamidinemethanesulfonate.
 5. A preventive or therapeutic agent for osteoporosis,according to claim 3, comprising the effective amount of the4-{5-[4-(5-isopropyl-2-methyl-1,3-thiazol-4-yl)phenoxy]pentoxy}-benzamidinehydrochloride.